Process for regenerating 2-chlorotrityl resins

ABSTRACT

The present invention relates to a recycling process for the preparation of solid phase bonded 2-chlorotrityl chloride (2-CTC resin) useful for solid phase peptide synthesis, wherein active chloride CTC resin is regenerated by reacting the spent resin with a chlorinating agent in organic solvent.

BACKGROUND OF THE INVENTION

Trityl resins are useful solid phase bonded protecting groups,originally developed for solid phase peptide synthesis and as well usedfor solid phase organic synthesis. Solid phase bonded 2-chlorotritylchloride (2-CTC resin) is commercial available [CBL Patras] and based oncrosslinked polystyrene (Ps) or modified polystyrenes (e. g. tentagel,polystyrene grafted with polyethyleneglycol) [1: catalog ACT,Noviabiochem]. The 2-CTC linker is an acid labile resin and because ofits excellence properties playing a significant role in novel linkertechnology [2: THL 1997, 6347, 2629; Int. J. Pep. Prot. Res. 37 (1991)513]. Cleavage of peptides from 2-CTC resin can be effected by treatmentunder very mild acidic conditions (e. g. dilute TFA/DCM, AcOH/DCM, HFIP)[1, 3: Liebigs A. 1988, 1079, THL 1997, 7299]. Despite being anexpensive and a valuable synthetic tool, a simple, efficient, gentle,reproducible experimental description for the recycling of—in solidphase synthesis—“used 2-CTC resin” without any loss of activity is sofar unknown in the literature.

To synthesize 2-CTC resin from the 2-chlorotriphenylcarbinol resin(2-CT-OH resin), references are given to the experimental procedure ofthe parent tritylchloride resin from the triphenylmethanol resin in ananalogous manner [4: THL 1989, 3943; Can. J. Chem. 54 (1976) 926] ormore detailed [1, 5: THL 1998, 3241; 6: React. Funct. Polym. 41 (1999)111].

WO 01/85758 A2 [7] discloses the recycling of carboxylic acid (e.g.preferably trifluoroacedic acid) trityl ester resins with HCl in CH₂Cl₂.Apart of the fact that trifluoroacedic acid trityl esters are subject tohydrolysis [8: Z. Phy. Chem. 113 (1978) 199] and therefore difficult tohandle, the reexamination of the example given in [7] achieved only 77%of the content of active chloride [6] before use.

After loading of 2-CTC resin with Fmoc-amino acids, unreacted activesites are end-capped with alcohols, especially methanol and Fmocprotecting groups are deprotected with amines, especially piperidine [1,9: W. C. Chan, P. D. White in Fmoc Solid Phase Peptide Synthesis]leading to a certain amount of methylether and amine substructures inthe resin. These substructures decrease the yield of the recycling stepand consequently the activity (content of active chloride) of therecycled resin.

SUMMARY OF THE INVENTION

Surprisingly it has now been found, that by carrying out a reaction ofused 2-CTC resin (comprising of a compound of formula II) in thepresence of a chlorinating agent and an organic solvent, the activechloride content of the recovered 2-CTC resin can be successfullyregenerated. Further it has been found that such recycling of “usedresin” takes place approximately quantitatively due to conversion ofcompound of formula II wherein R is as defined below to 2-CTC resin. Therecycled 2-CTC resin loaded successful Fmoc-Trp(Boc) or Fmoc-Leu [9: p.217]. It was found that chlorination with HCl in the presence of anorganic solvent is also applicable for the conversion of 2-CT-OH resinto 2-CTC resin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes a process for the preparation of solidphase bonded 2-chlorotrityl chloride (2-CTC resin) of formula I

-   wherein Ps is a polymeric support and n has the following meaning:    1≧n>0 comprising the reaction of solid phase bonded 2-chlorotrityl    of formula II

-   wherein R is OH or/and OC₁₋₄-alkyl or/and NR′R″ (i.e. the resin as a    whole may have bound to it any one of or a combination of such    groups),-   wherein R′ and R″ independently of each other represent C₁₋₄-alkyl,    or R′ and R″ together with the nitrogen to which they are bonded    represent a 5 to 8 membered heterocyclic radical-   in the presence of a chlorinating agent and an organic solvent.

In further preferred embodiments the reaction is carried out wherein

-   R is OH, OCH₃, OC₂H₅, OC₃H₇, or-   R′ and R″ independently of each other represent methyl, ethyl, or-   R′ and R″ together with the nitrogen to which they are bonded    represent piperidine.

Most preferred is the case wherein R is OH or/and OCH₃ or/and piperidineand the used resin is reacted in the presence of HCl and an organicsolvent.

The term “polymeric support” within the present invention meanspolymeric resins suitable for use in solid phase synthesis, or surfacesin which polymers, having synthesis properties similar to thesepolymeric resins are attached to a solid support, or modified silicagels suitable for use in solid phase synthesis, preferably polystyrene(0-25% divinylbenzene crosslinked), more preferred 0-10% divinylbenzenecrosslinked and most preferred 1% divinylbenzene crosslinked.

The term “solid phase bonded 2-chlorotrityl” within the presentinvention means 2-chlorotrityl bonded to the polymeric support asdefined above.

The value “n” within the present invention has the following meaning:1≧n>0, therefore the value “n” may be between 0 and 1, whereas 1 isincluded. In a preferred embodiment, the value “n” has the followingmeaning: 0.9≧n≧0.5.

The term “alkyl” as used herein denotes an optionally substitutedstraight or branched chain hydrocarbon residue containing 1 to 4 carbonatoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, 1-sec-butyl,isobutyl and tert.-butyl. Preferably, the term “alkyl” denotes anoptionally substituted straight or branched chain hydrocarbon residuecontaining 1 to 3 carbon atoms. More preferred the term “alkyl” denotesa methyl or ethyl group. Most preferred the term “alkyl” denotes amethyl group.

Within the present invention a compound of formula II wherein R is OHor/and OC₁₋₄-alkyl or/and NR′R″ wherein R′ and R″ independently of eachother represent C₁₋₄-alkyl, or R′ and R″ together with the nitrogen towhich they are bonded represent a 5 to 8 membered heterocyclic radical(preferably wherein R is OH, OCH₃, OC₂H₅, OC₃H₇, dimethylamine,diethylamine, methylethylamine or/and piperidine; most preferred whereinR is OH or/and OCH₃ or/and piperidine) is used.

The ratio of OH and OC₁₋₄-alkyl and NR′R″ maybe between 100:0:0 and0:100:0 and 0:0:100. In a preferred embodiment the compound of formulaII wherein R is OH is enriched.

Within the present invention 5 to 8 membered heterocyclic radicals forthe term NR′R″ are the following moieties: pyrrolidinyl, piperidinyl.

According to the present invention, “used resin” (formula II), obtainedafter peptide cleavage, e.g. in the case of the polypeptides T-20 orT-1249 [10: U.S. Pat. No. 5,464,933, 12: U.S. Pat. No. 6,258,782] andquenching with pyridine (traces of water) [13: JACS 80 (1958) 812], isrecycled after washing (e.g. dioxane) in the presence of a chlorinatingagent and an organic solvent. The carbinol substructure in “used resin”can be verified by IR-spectroscopy (ν_(OH)=3566 cm⁻¹). Trace amounts ofMethanol (evidence of minimum —OCH₃ content in “used resin”) can bedetected by GC in the filtrate of the recycling experiments and nitrogencontent by elemental analysis of “used resin”. The recycling reactionmay be carried out in organic solvents: e. g. toluene, chlorobenzene,CH₂Cl₂, DMSO, NMP (1-metyl-2-pyrrolidinone), DMF, ethers or cyclicethers with high absorption capacity of HCl gas (such as alkylethers,DME, Diglyme, THF or dioxane). The recycling reaction is preferablycarried out in the presence of THF or dioxane, most preferred in thepresence of dioxane.

Further, the recycling reaction may be carried out with chlorinatingagents known from textbooks about organic chemistry (e.g. from J. March,“Advanced Organic Chemistry: Reactions, Mechanisms, and Structure”,4^(th) ed. John Wiley & Sons(1992)) such as PCl₅, PCl₃, POCl₃, SOCl₂,CH₃COCl, CO₂Cl₂, (CH₃)₃SiCl or HCl. Preferred chlorinating agents arePCl₅, PCl₃, POCl₃ or HCl, and most preferred chlorinating agent is HCl.

In a further embodiment, the present invention maybe carried out in asingle percolation or in repetitive percolations.

In a preferred embodiment, the process is carried out with excess HCl.

The reaction may be carried out at a temperature between 0° C. and 110°C. preferably at a temperature between 0-50° C. and most preferred at atemperature between 10° C. and 25° C.

The reaction may be carried out in the organic solvent (preferreddioxane or THF, most preferred dioxane) wherein the HCl content is about10-20 g/100 ml, most preferred >20 g/100 ml.

In a further embodiment of the invention, the reaction may be carriedout in the organic solvent (preferred dioxane or THF, most preferreddioxane) wherein the HCl content is >0 to about 40 g/100 ml, preferably10-35 g/100 ml, and most preferred 20-30 g/100 ml.

The reaction may be carried out for 6-96 h preferably for 17-96 h andmost preferred for 21-24 h.

EXAMPLES

Procedure:

In a double-walled jacket reactor with glass frit bottom 5 g pure “usedresin” (formula II) was—after pretreatment in the organic solvent andsuction of the solvent—stirred slowly in the organic solvent withdissolved chlorinating agent at specified reaction temperature (T) forspecified reaction time (t). After completion of the percolation andsuction of the reaction solution the remaining resin was washed with thereaction solvent, treated with hexane and dried under vacuum at 35° C.The content of active chloride of the recycled 2-CTC resin (formula I)was determined [6] and its content of nitrogen by elemental analysis wasbelow detection limit. In the examples set forth in Table 1, the virgin2-CTC resin (formula I) had content of active chloride before use: ˜1.27mol/kg. The “used resin” (formula II) had no active chloride before useand the content of compound of formula II wherein R is OC₁₋₄-alkyl isbetween 0 and 5%, and the content of nitrogen is between 0 and 0.2%.

TABLE 1 formular II content of active R is solvent chlorinating agent T[° C.] t [h] chloride [mol/kg] OH, OCH₃, 80 ml toluene 10 ml PCl₃ 25 170.76 piperidine OH, OCH₃, 80 ml toluene 10 ml PCl₃ 50 17 0.75 piperidineOH, OCH₃, 80 ml toluene 10 ml POCl₃ 25 17 0.62 piperidine OH, OCH₃, 80ml toluene 10 ml SOCl₂ 25 17 0.97 piperidine OH, OCH₃, 80 ml toluene 10ml SOCl₂ 110 15 1.02 piperidine OH, OCH₃, 80 ml toluene 10 ml CH₃COCl 2517 0.39 piperidine OH, OCH₃, 80 ml toluene 10 ml CH₃COCl 50 17 1.03piperidine OH, OCH₃, 200 ml toluene 10 g PCl₅ 110 17 0.77 piperidine OH,OCH₃, 120 ml CH₂Cl₂ 10 g PCl₅ 25 17 1.22 piperidine OH, OCH₃, 80 mlCH₂Cl₂ 10 ml SOCl₂ 40 65 0.97 piperidine OH, OCH₃, 40 ml dioxane 9.8 gHCl 25 24 1.22 piperidine (24.5 g/100 ml) OH, OCH₃, 50 ml dioxane 8.7 gHCl 25 24 1.29 piperidine (17.4 g/100 ml) OH, OCH₃, 71 ml dioxane 11.1 gHCl 25 17 1.14 piperidine (15.6 g/100 ml) OH, OCH₃, 90 ml dioxane 9.2 gHCl 25 24 1.21 piperidine (10.2 g/100 ml) OH, OCH₃, 90 ml dioxane 14 gHCl 25 24 1.23 piperidine (15.6 g/100 ml) OH, OCH₃, 90 ml dioxane 14 gHCl 10 24 1.23 piperidine (15.6 g/100 ml) OH,OCH₃, 90 ml dioxane 14 gHCl 25 65 1.18 piperidine (15.6 g/100 ml) OH, OCH₃, 2x 90 ml dioxane 2 ×14 g HCl 25 2x 24 1.16 piperidine (15.6 g/100 ml) OH, OCH₃, 90 mldioxane 14 g HCl 25 20 1.21 piperidine (no pretreatment) (15.6 g/100 ml)OH, OCH₃, 90 ml dioxane 24.3 g HCl 25 21 1.29 piperidine (27 g/100 ml)OH, OCH₃, 90 ml dioxane 24.3 g HCl 25 20 1.27 OC₂H₅, (27 g/100 ml)piperidine OH, OCH₃, 90 ml dioxane 23 g HCl 25 22 1.31 i-OC₃H₇ (25.5g/100 ml) OH 90 ml dioxane 18.9 g HCl 25 24 1.6 (21 g/100 ml) OH 90 mlTHF 19.7 g HCl 25 18 0.99 (21.9 g/100 ml) OH 90 ml THF 31.1 g HCl 25 241.15 (34.5 g/HCl)

From the data presented in Table 1, it is clear that by carrying out thereaction according to the present invention active chloride issubstantially regenerated in the recovered 2-CTC resin.

1. A process for the preparation of solid phase bonded 2-chlorotritylchloride (2-CTC resin) of formula I

wherein Ps is a polymeric support and n has the following meaning: 1≧n>0comprising the reaction of solid phase bonded 2-chlorotrityl of formulaII

wherein R is OH or/and OC₁₋₄-alkyl or/and NR′R″ wherein R′ and R″independently of each other represent C₁₋₄-alkyl, or R′ and R″ togetherwith the nitrogen to which they are bonded represent a 5 to 8 memberedheterocyclic radical in the presence of a HCl and dioxane.
 2. Theprocess according to claims 1 wherein n is 0.9≧n≧0.5.
 3. The processaccording to claim 1 wherein the reaction temperature is between 0 and110° C.
 4. The process according to claim 1 wherein the HCl content indioxane is >0 to about 40 g/100 ml.
 5. The process according to claim 1wherein the reaction time is 6-96 h.
 6. The process according to claim 1wherein it is carried out in a single percolation or in repetitivepercolations.